PROJECT SUMMARY The islets of Langerhans are essential regulators of glucose homeostasis. Loss of islet function, particularly ?- cell function, leads to diabetes. Islet transplantation and regeneration represent the most promising strategies to treat diabetes. The majority of research effort is spent studying ?-cells, but islets are composed of multiple endocrine cell types (principally ?-cells, ?-cells, ?-cells and ?-cells) that function together to effectively regulate blood glucose. Growing evidence suggests that the physical organization of these different cell types, and the resulting juxtacrine and paracrine signaling, is instrumental in the resulting homeostatic behavior. Interestingly, both the proportion and 3D arrangement of endocrine cells within an individual islet is highly variable even among closely related species. Given the variability of the normoglycemic set point between species, these observations imply that islet structure is intricately linked to functional output. However, the molecular tools to directly engineer islet cells with user-defined 3D cytoarchitectures have not been developed. We are in a unique position to systematically understand the relationship between micro-organ structure and function because of new tools that we have been developing to engineer multicellular self-assembly and self-organization (17,18). A particularly exciting new class of tools are synthetic adhesion molecules (synCAMs). synCAMs enable programmable cell- cell interactions by fusing engineered hetero- or homo-typic extracellular interaction domains to the transmembrane and intracellular domains of cell adhesion molecules. This synthetic morphological toolkit allows us to link cells together is highly specified patterns or clusters. The overall goal of this proposal is to ask whether we can engineer multicellular organization of islets to determine the principles of how cytoarchitecture determines homeostatic function. By elucidating a set of design principles that govern islet function, we believe that customizable or tunable pseudoislets could be engineered to adopt optimized behaviors that might better treat diabetic patients. In Specific Aim 1, we will establish the expression and function of synCAMs in PSC- derived islet cells using high-throughput confocal microscopy. In Specific Aim 2, we will systemically probe the relationship between pseudoislet cellular composition and cytoarchitecture on the metabolic maturation of PSC- derived pseudoislets by coupling high-throughput confocal microscopy and automated 3D image analysis to in vitro metabolic flux assays. In Specific Aim 3, we will use our synCAM tools to explore the effect of tuning cell- cell interactions, the overall size and the relative ratio of endocrine cells within a pseudoislet on glucose homeostasis function in vitro and in vivo. This fellowship will provide me with the experience and tools critical for my goal of developing into a productive, independent scientist. My sponsor, Dr. Lim, will provide the necessary expertise in synthetic biology and optimizing synthetic cellular control modules to properly mentor me in this work. We believe our approach is innovative and could have a broader impact on the field of islet transplantation and treatment of diabetes in addition to equipping me with the training to support my career goals.